The Egyptian Journal of Hospital Medicine Vol., 22 : 60 - 72 ... National Research Center, Dokki, Cairo, Egypt. Abstract ... Americans use herbal remedies (Astin,.
The Egyptian Journal of Hospital Medicine Vol., 22 : 60 - 72
March 2006 I.S.S.N: 12084 1687 - 2002
Antioxidant effect of parsley and panax ginseng extract standardized with ginsenosides Rg3 against alteration induced in reproductive functions in male mice Aziza M. Hassan1 and Mosaad A. Abdel-Wahhab2 1 Cell Biology Dept. and 2Food Toxicology & Contaminants Dept. National Research Center, Dokki, Cairo, Egypt Abstract In the present study, the antigcidant effects of parsley oil and panax ginseng have been evaluated against the clastogenecity of ZEN. One hundred and eight mature male mice were distributed into nine treatment groups, including the control group and the groups treated with parsley oil (0.6 ml/kg b.w), panax ginseng extract (40 mg/kg b.w) or parsley oil plus panax ginseng extract with or without ZEN (10 µg/kg b.w). Animals within different treatment groups were divided into two subgroups (A and B). Subgroup A were used for the determination of serum testosterone levels and chromosomal aberrations and received their respective doses for two weeks whereas, subgroup B were used for sperm abnormality and received their respective doses twice a day for one week and sacrificed after 30 days. The results indicated that ZEN treatment resulted in a significant decrease in testosterone concentration, sperm count and sperm motility. Whereas it caused a significant increase in abnormal sperms counts and total chromosomal aberrations in germ cells. Animals treated with parsley oil or panax ginseng extract alone or in combination were comparable to the controls regarding all the tested parameters. The combined treatment with ZEN and parsley oil, panax ginseng or parsley oil plus panax ginseng extract resulted in a significant improvement in all tested parameters. Moreover, parsley oil was found to be effective than panax ginseng extract and the combined treatment was more effective than the single treatment. It could be concluded that both parsley oil and panax ginseng extract induced a protective action against ZEN-induced alteration in the reproductive performance and the combined treatment may be useful than the single treatment. Key words: Zearalenone, parsley oil, panax ginseng, cytogenetic, germ cells, testis, testosterone and chromosomal aberration.
Introduction Herbal remedies are used world wide to alleviate symptoms, to treat illnesses, and to promote overall wellness. An estimated 60% of the world's population and 40% of Americans use herbal remedies (Astin, 1998). However, use of medicinal herbs may be unsupervised and unproved efficacy (Boniel and Dannon 2001; Ernst and Cassileth 1999). Parsley (Petroselinum crispum) is an important culinary herb native to the Mediterranean area. It has been cultivated
for more than 2,000 years. Parsley is a member of the Umbelliferae family that has been employed in the food, pharmaceutical, perfume, and cosmetics industries (Lopez et al., 1999). It is widely distributed in Egypt and grown in gardens and fields. Parsley has been reported to have a number of possible medicinal attributes including, antimicrobial, (Wong and Kitts 2006) antianemic, menorrhagic, (Baytop, 1984) anticoagulant, antihyperlipidemic, antihepatotoxic, (Ozturk et al., 1991) antioxidant,
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(Nielsen et al., 1999) and laxative (Kreydiyyeh et al., 2001). It has been used to treat lumbago, as a blood pressure regulator, to treat eczema, knee, ache, impotence and nose bleed (Manderfeld et al., 1997). Parsley seed are also used as a diuretic and the hypoglycemic activity of parsley has been shown by Ozsoy et al., (2006). The constituents of parsley which include ascorbic acid, carotenoids, flavonoids, coumarins, apiole, various terpenoic compounds, phenyl propanoids, phthalides, furano coumarins, and tocopherol, have been chemically investigated (Tunali et al., 1999). Components of fresh parsley leaf scavenge superoxide anion in vitro (Campanella et al., 2003), and the methanol extracts of parsley scavenge hydroxyl radical in addition to protecting against ascorbic acid induced membrane oxidation (Fejes et al., 2000). Supplementation of diets with fresh parsley leaf can increase antioxidant capacity of rat plasma (Hempel et al., 1999). Ginseng is one of the most popular medicinal plants, the roots of which have long been traditionally used for strengthening immunity, providing nutrition and recovering health from fatigue (Blumenthal, 2000). The pharmaceutical activities of ginseng roots have been proven recently by many investigators, and ginseng has become the famous medicinal plant all over the world. Ginseng roots contain various pharmaceutical components ginsenosides (saponins), polyacetylenes, polyphenolic compounds and acidic polysaccharides, and among the components, ginsenosides are the most pharmaceutically active (Kim et al., 2005). Until now, 38 ginsenosides have been isolated from ginseng roots, with five major ginsenosides (ginsenosides Rb1, Rb2, Rc, Re and Rg1) constituting more than 80% of the total ginsenosides (Kim et al, 1999). Moreover, ginseng intake was associated with a decreased risk for most cancers including carcinomas of the esophagus, stomach, colon, pancreas, lung and liver (Matsuda, 1986; Jeong et al,. 1997; Dayan and Paine 2001). In the recent years, many studies have aimed to convert major ginsenosides to the more active minor ginsenosides using heating, acid
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treatment, enzymatic and microbial conversion (Kim et al., 2005). The minor ginsenoside, Rd, has been known to enhance the differentiation of neural stem cells, while other ginsenosides induce no differentiation of neurons (Shi et al., 2005) and are known to protect neural systems against neurotoxicity by attenuating NO overproduction (Choi et al., 2003, Mannaa et al., 2006). The pharmaceutical property of ginseng roots in protecting neurons from neurotoxic chemicals, such as kainic acid, is attributed mostly to ginsenoside Rd (Lee et al., 2003). Ginsenoside Rd has been known to protect kidney from apoptosis and DNA fragmentation caused by chemical drugs and cancer drugs ( Yokozawa and Owada, 1999; Yokozawa and Dong, 2001, Mannaa et al., 2006). Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin present in corn, as well as food mixture for farm animals (Hagler et al., 1984; Molto et al., 1997; Placinta et al., 1999). Studies in various species (rodents, pigs and monkeys) have shown that ZEN and its metabolites have estrogenic and anabolic activities (Etienne and Dourmad 1994) and it has cytotoxic effects to mammalian cell cultures (Cetin and Bullerman, 2005). ZEN was associated with hyperestrogenism and several physiological alterations of the reproductive tract in several laboratory animals (mice, rat, guinea-pig, hamster, rabbits) (Creppy, 2002). ZEN ingestion through contaminated feed is associated with decreased reproductive capacity and other hyperestrogenic conditions, such as vaginal swelling, enlargement of mammary glands, and testicular atrophy in farm animals (Wannemacher et al., 2000). The strong estrogenic effect of ZEN is due to its competition with 17 Bestradiol in the binding to cytosolic estrogen receptors present in the uterus, mammary gland, hypothalamus and pituitary gland (Kuiper-Goodman et al., 1987; Creppy, 2002). Several toxic effects of ZEN were demonstrated, it induces apoptosis, DNA fragmentation (Kim et al., 2003; Abid-Essafi et al., 2003), micronuclei production (Ouanes et al., 2003), chromos-ome aberration (IARC, 1993; Ouanes et al., 2005) and DNA-adduct formation (Pfohl-
Antioxidant effects of parsley and panax ginseng......................
Leszkowicz et al., 1995). These toxic effects are unlikely to be due to the estrogenic activity of ZEN. Several processes are known to play a role in the molecular events leading to cell damage. The carcinogenicity of ZEN is still questionable since it is classified by IARC in group 3, i.e. not classifiable as to its carcinogenicity to humans (IARC, 1993; Yosida and Amano, 1965). The objectives of the current study were to evaluate the potential of panax ginseng extract and parsley oil against the cytotoxic effects and the reproductive disorders induced by ZEN in male mice.
Materials And Methods Chemicals: ZEN standards was purchased form Sigma Chemical Co. (St. Louis, MO). Testosterone Kit was purchased from ELISA (Enzyme Linked Immuno System Assay)., Italy. All other chemical were of the highest purity commercial available. Parsley oil were purchased from ElCaptain Company (CAP PHARM), 6th October, Egypt. Panax ginseng: The standardized Panax ginseng extract EFLA400 (Phoenix ginseng) (Batch No. 303298) of Panax ginseng C. A. Mayer was prepared according to the published procedure (Korean patent 0425022, PCT/KR2003/000003) and was supplied from Lotte Group R & D Centre (Seoul, Korea). The content of ginsenoside Rg3, a pharmacologically active ingredient of Phoenix ginseng, was 3.6% (w/w) as determined by HPLC. Animals: A total of 108 adult male Swiss albino mice (eight-week old, weighing 25 ± 2 gm) purchased from Animal House Colony, NRC Dokki, Giza, Egypt. Animals were maintained on standard laboratory diet (protein, 16.04%; fat, 3.63%; fiber, 4.1%; and metabolic energy, 0.012 MJ) and water ad libitum. After an acclimation period of 1 week, animals were divided into nine groups (12 mice/ group) and housed in filtertop polycarbonate cages (six animals/cage) the animals were maintained in an environmentally standard controlled.
Experimental design: Animals within different treatment groups were treated as follow: group 1 untreated control; group 2, treated orally with corn oil; group 3, treated orally with Korean panax ginseng extract (40 mg/kg b.w) suspended in water; group 4, treated orally with parsley oil (0.6 ml/kg b.w); group 5, treated orally with panax ginseng extract plus parsley oil; group 6, treated orally with ZEN (10 µg/kg b.w) in corn oil; group 7, treated orally with ZEN plus panax ginseng extract; group 8, treated orally with ZEN plus parsley oil; and group 9, treated orally with ZEN plus panax ginseng extract and parsley oil. All groups were divided into two subgroups A and B. Animals in subgroup A (within all treatment groups) were used for the determination of blood serum testosterone level and chromosomes analysis of germ cells and treated with the respective treatment for 15 days. On day 16 blood samples were collected for testosterone determination then animals were sacrificed by cervical dislocation and testes were removed for chromosomes analysis of germ cells. Whereas animals in subgroup B were used for the determination of sperm abnormality and treated twice/day with their respective doses for one week and sacrificed after 30 days and epididymis were removed for the sperm abnormality study. Chromosome analysis of germ cells was carried out according to the method described by Evans et al., (1964) and modified by Russo (2000). For testosterone determination, blood samples were collected from all animals in subgroup A from retro-orbital venous plexus (Schermer, 1967) then left to clot, centrifuged at 5000 rpm under cooling for 10 min and serum was separated for testosterone determination by enzyme linked immunoassay procedure as described by Parker (1981). The technique of Wyrobek et al., (1984) was adopted for sperm abnormality study with minor modifications. Epididymis (free of fats, vas deferens and other tissues) from each side of testis of either control or treated mice was dissected out and the inner content squeezed out into 10 ml of 0.87 % normal wormed saline separately. The content was thoroughly shaken, filtered
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through a silken cloth and dropped on grease-free clean slides to determine the movement and swimming ability of sperm (motility) using microscope. Spermatozoa were counted using heamocytometer and a drop of a heamogenate smeared on a cleaned slide, allowed to air dry and stained by Eosin Yellow to determine the head and tail abnormality of sperms. Statistical Analysis: Students't-test was conducted as per standard procedure for determining the significance level of the differences between treated and control data according to Parker, (1979).
Results The results of the cytogenetic study (Table 1) revealed that treatment with ZEN resulted in a significant increase in frequency of aberrations in spermatocytes (19.0 ± 1.0) whereas, animals treated with either parsley or ginseng extract were nearly as the control groups. Cotreatment with parsley or ginseng extract plus ZEN showed a significant decrease (p
